A COMPARATIVE  STUDY  OF  THE  HYDROLYSIS 
OF  CERTAIN  ESTERS  BY  LIPASE 


By 

ELIZABETH  CHARLOTTE  HYDE 

B.  A.  Mt.  Holyoke  College,  1909 


THESIS 


SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS 
FOR  THE  DEGREE  OF  MASTER  OF  SCIENCE  IN  CHEMISTRY 
IN  THE  GRADUATE  SCHOOL  OF  THE 
UNIVERSITY  OF  ILLINOIS,  1922 


URBANA,  ILLINOIS 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/comparativestudyOOhyde 


UNIVERSITY  OF  ILLINOIS 
THE  GRADUATE  SCHOOL 


.,ar  23__  .192  ° 


I HEREBY  RECOMMEND  THAT  THE  THESIS  PREPARED  UNDER  MY 

SUPERVISION  BY Izajaal ii.  Chari ott e I£.dj3 

ttxttttt .ff>  A COMPARATIVE  STUDY  Off  THE  HYDROLYSIS  OF  CBRMIIL 

ESTERS  BY  LIPASE 


BE  ACCEPTED  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR 
THE  DEGREE  OF ...ASTER  OF  SCIENCE 


Recommendation  concurred  in* 


Committee 


on 


Final  Examination* 


^Required  for  doctor’s  degree  but  not  for  master’s 


499016 


■ I 


The  writer  is  glad  to  have  this 
opportunity  to  acknowledge  her  indebted- 
ness to  Dr.  Howard  B • Lewis  for  his  kind, 
assistance  in  this  research. 


TABLE  OS’  CONTENTS 


I GENERAL  INTRODUCTION 1 

II  HISTORICAL  ...  .....  2 

III  EXPERIMENTAL,  PART  I .....  4 

IV  EXPERIMENTAL,  PART  II 15 

V SUMMARY 32 

VI  BIBLIOGRAPHY 34 


I INTRODUCTION 


Enzymic  reactions  are  important  in  physiological  processes. 
It  is  possible  to  trace  most  processes  of  anabolism  and  catabol- 
ism to  enzyme  action,  but  there  our  knowledge  ceases  because 
the  nature  and  mode  of  the  action  of  enzymes  is  not  thoroughly 
understood.  To  explain  physiological  processes  from  a chemical 
standpoint  it  is  necessary  to  understand  the  kinetics  of  the 
various  enzyme  reactions.  It  has  been  the  ©eject  of  this  study 
to  investigate  under  carefully  controlled  conditions  the  mode 
of  action  of  the  fat-splitting  enzymes  or  lipasis  on  certain 
types  of  esters.  The  results  reported  here  are  concerned,  with 
the  action  of  liver  lipase  on  certain  esters  of  the  dicarboxy- 
lic  acid 3. 


II  HISTORICAL 

Certain  facts  have  been  obtained  regarding  the  phenomena  j 
of  the  hydrolytic  action  of  lipase  021  esters  of  dicarboxylic  j 
acids.  Kastle(l)  in  1902  found  that  diethyl  succinate  was  read-' 
ily  hydrolysed  by  liver  lipase  but  he  offered  no  quantitative 
data  as  to  the  extent  or  products  of  hydrolysis.  He  also  ob- 
served that  the  metallic  salts  of  the  monoethyl  esters  such  as 
potassium  ethyl  succinate  and  sodium  ethyl  succinate  were  not 
split  by  lipase.  The  inactivity  of  lipase  toward  this  class  of 
compounds  was  explained  by  Kastle  as  being  due  go  the  ionization 
of  the  metallic  salts.  He  believes  that  ionized  compounds  are 
stable  toward  lipase. 

In  1903  Doyen  and  Morel(2  ) observed  that  serum  lipase  could 
hydrolyse  diethyl  succinate.  However  they  did  not  isolate  the 
products  of  hydrolysis. 

Morel  and  Terronie(  5 ) undertook  a comparative  study  of  the 
hydrolysis  of  the  diethyl  esters  of  malonic,  succinic,  glutanic, 
suberic  and  sebacic  acids  by  the  pancreatic  juice  of  the  dog 
and  concluded  that  the  ease  of  hydrolysis  increased  with  the 
increase  of  the  molecular  weight  of  the  acid  up  to  glutaric 
acid.  The  maximum  hydrolysis  obtained  by  them  for  diethyl  mal- 
onate  and  diethyl  succinate  were  2.6  per  cent  and  12.9  per  cent 
respect ively • 

Christman  and  Lewis(6)  made  a careful  study  of  the  action 


of  liver  lipase  on  various  esters,  including  ethyl  succinate  and 


- 


/ 


I * 


-3- 


ethyl  malonate.  Kastle's  observation  regarding  the  stability 
of  the  metallic  salts  of  the  monoethyl  esters  of  the  dicarboxy- 
lic  acids  was  confirmed  by  their  experimentation.  Their  results 
also  seem  to  snow  that  the  hydrolysis  of  the  diethyl  esters  of 
malonic  and  succinic  acids  can  proceed  only  to  the  removal  of 
one  ethyl  group  and  that  the  monoethyl  esters  as  well  as  their  ! 
metallic  salts  are  relatively  stable  toward  lipase. 


-4™ 


ITT  EXPERIMENTAL 

Lipase  may  act  in  three  ways  in  the  hydrolysis  of  an  ester 
of  a dicar'box3rlic  acid.  One  ethyl  group  my  be  removed  and 
the  second  ethyl  group  left  intact.  One  ethyl  group  may  be 
removed  first  and  the  second  ethyl  group  subsequently  attacked, 
or  both  ethyl  groups  may  be  removed  simultaneously  by  the 
lipase.  It  seems  quite  probable  that  lipase  attacks  different 
esters  in  a different  manner. 

To  determine  the  mode  of  lipolytic  hydrolysis  it  is  nec- 
essary to  find  out  what  products  result  from  the  reaction. 

This  research  has  been  divided  into  two  main  problems,  the 
first  being  a study  of  the  time  required  for  the  diethyl  esters 
of  dicarboxylic  acids  to  attain  an  equilibrium  in  their  hydro- 
lytic reaction  with  liver  lipase  under  varying  conditions  of 
acidity,  and  the  second  an  attempt  at  the  isolation  of  the 
products  of  these  reactions. 

The  esters  studied  in  this  manner  were  diethyl  malonate, 
diethyl  succinate,  diethyl  glutarate  and  diethyl  adipate.  A 
study  of  the  hydrolysis  of  ethyl  propinnate  by  liver  lipase 
was  made  in  order  co  determine  to  what  extent  the  hydrolysis 
of  a simple  ester  could  be  effected  under  the  conditions  of 
these  experiments. 

The  lipase  extract  which  was  used  in  this  research  was 
prepared  from  a liver  taken  from  a hog  immediately  after  the 
death  of  the  animal.  According  to  the  method  of  Kanitz( 3 ) 


-5- 

two  parts  of  glycerol  were  added  to  one  part  of  the  finely  cnop- 
ped  tissue.  After  a thorough  shaking  the  mixture  was  allowed 
to  stand  at  least  a week  and  then  filtered  through  cheesecloth 
when  needed.  The  extract  retained  its  constant  lipolytic  action 
unimpaired  for  several  months. 

Trie  diethyl  propionate,  diethyl  malonate,  and  diethyl  succin- 
ate used  were  commercial  products.  The  diethyl  glutarate  was 
made  in  the  organic  laboratory  while  the  diethyl  adipate  was 
prepared  from  adipic  acid  made  by  the  method  of  Bouvealt  and 
Locquin(4)  in  the  laboratory  in  which  this  research  was  carried 
on.  The  purity  of  the  esters  used  was  determined  by  means  of 
saponification  values.  To  obtain  optimum  conditions  for  the 
action  of  lipase  it  is  necessary  that  the  concentration  of  the 
ester  be  such  that  complete  solution  is  effected,  therefore 

■JT 

^T,  and  concentrations  were  used. 

5cc  portions  of  the  glycerol  extract  were  measured  by  means 
of  a pipette  into  200  cc  Erlenmeyer  flasks.  To  each  flask  was 
added  25  cc  of  the  standard  ester  solution.  These  flasks  were  . 
incubated  at  room  temperature  for  varying  periods  of  time.  The 
acidity  developed  by  the  reaction  was  then  titrated  with  ^ 
NaOH,  using  phenolthaiein  as  an  indicator.  The  flasks  were  ar- 
ranged in  pairs  and  one  pair  was  titrated  after  15  minutes  and 
a second  pair  after  minutes,  etc.  Each  pair  of  flasks  was 
also  retitrated  at  the  intervals  shown  in  the  tables.  The  fig- 
ures given  in  the  tables  represent  the  total  volume  of  Ha OH 

required  for  neutralization  and  are  the  averages  of  check  deter- 


-6- 

minations  from  which  the  blanks  due  to  the  acidity  of  the  ester 
solution  and  lipase  extract  have  been  subtracted.  The  total 
acidity  of  the  blank  was  at  no  time  equivalent  to  more  than  .3 
cc  of  yy  II  a OH.  The  left  hand  figures  in  each  line  of  the 
tables  represents  the  average  acidity  developed  in  two  flasks 
which  were  first  titrated  at  the  end  of  the  time  stated  at  the 
head  of  the  column  in  which  the  figures  are  found.  All  figures 
to  the  right  represent  the  total  acidity  which  had  developed 
during  the  interval  of  time  stated  at  the  top  of  the  column  in 
which  the  figures  are  found. 


TABLE  I 

Hydrolysis  of  0.025  F ethyl  propionate  by  lipase.  25  cc  por- 
tions of  ester  were  used.  Bor  the  complete  saponification  of 
this  amount  of  ester  5*3  cc  of  0,09872  F FaOH  were  required. 


Time 

30’  1 hr 

2 hr  e 

3 hrs 

4 hrs 

9 hrs 

6 hrs 

10  hrs 

11  hrs 

Standard  FaOH 

required  for 

neutralizat ion 

of  acidity 

3-05  4.7 

5.05 

5.O7 

5.O7 

5.07 

5.07 

5.07 

5.07 

3,72 

4.7 

4.8 

4,8 

4.8 

4,8 

4.8 

4.8 

4.27 

4,45 

4.55 

4.59 

4.55 

4.55 

4.55 

4.35 

4.55 

4.95 

4.55 

4.55 

4.55 

4.92 

9.17 

5.17 

5.17 

5.17 

4.97 


5.02 

5.09 


5.02 

5.05 

5.00 


5.02 
5.05 
5,00 
5 .15 


—3- 


TABLE  II 

Hydrolysis  of  0,05  IT  diethyl  malonate  by  lipase.  25  cc  portions) 
of  ester  were  used.  For  trie  complete  saponif ication  of  this 


amount 

of  e 

ster  12 

.6  c c 

0 

•b 

0 

• 

09872 

IT  Ha  OH 

were 

required . 

Time 

30’ 

1 hr 

2 hrs 

3 hrs 

4 hrs 

5 hrs 

6 hrs 

7hrs 

8 hrs 

9 hrs 

Cone . 
of 

Ester 

2.0 

3.62 

5*52 

6.0 

6.25 

6. 3 

6.4 

6.4 

6.4 

6.4 

3*3 

5*57 

6.07 

6.22 

6.25 

6.25 

6.25 

6.25 

6*3 

4.75 

5*75 

6.1 

6.25 

6.25 

6.25 

6.25 

6,3 

5*20 

5.82 

6.12 

6.12 

6.12 

6.12 

6.12 

.05  H 

5*55 

6.02 

6.22 

6.55 

6.55 

6.4 

5.6? 

6.1? 

6.45 

6.47 

6.52 

5*3 

6.0 

6.3 

6.35 

5*9 

6.1 

6.1 

6.1 

Hydro 
29  ec 
tion 
were 

-9- 

TA3LE  III 

lysis  of  0,05  and  0.023  11  diethyl 
portions  of  ester  were  used.  For 
of  this  amount  of  ester  12.2  and  6 
respectively  required. 

succinate  hy  lipase, 
the  complete  saponifica- 
.00  cc  of  0.09872  N Ha OH 

L 

Time 

Cone  of 

30’ 

1 hr  2 hrs 

3 hrs 

4 hrs 

9 hrs 

7 hrs 

9 hrs  Ester  j 

Standard  Ha OH 

required  for 

neutralization 

of  acidity 

: 

2.67 

5-15  5.70 

9.72 

9.72 

S.72 

3 . 73 

5-75 

4.32  5.97 

6.02 

6.02 

6.02 

6.07 

6,07 

5.7 

6.02 

6.02 

6.02 

6.03 

6.09 

5.9 

6 . 09 

6.03 

6 . 03 

6,03  0.09  u 

6.07 

6.1 

6.1 

6.1 

6. '03 

6.19 

6.19 

6.13 

6.13 

6.2 

Time 

Cone,  of 

30» 

1 hr  2 hrs 

3 hrs 

4 hrs 

6 hrs 

8 hrs 

9 hrs  Ester 

Standard  Ha OH 

requir 

ed  for 

neutralization 

of  acidity 

1*95 

“2T73  ~1T 

2 

2,8 

2.8 

2,8 

273 — 1 

2.61  2.91 

2.91 

2.91 

2.91 

2,91 

2.91 

2.82 

2.8? 

2.87 

2.87 

2. 8? 

2.87 

2.9? 

2.97 

2,97 

2,97 

2.97  C.023  IT 

2.97 

2.97 

2.97 

2.97 

1 

2.9 

2,9 

2.9 

3.07 

3.07 

3*09 

VM 


-11- 

TABLE  V 

Hydrolysis  of  0,025  H solutions  of  diethyl  adipate  by  lipase. 


25  cc  portions  of  the  ester  soluttion  were  used, 
saponification  of  this  amount  of  ester  6.35  cc  0 
HaOH  were  required. 

Bor  complete 
f 0.09872  IT 

I 

Time 

30' 

1 hr 

2 

hrs 

3 hrs 

4 hrs 

5 hrs 

7 hrs 

9 hr 

s 11  hrs 

Standard 

NaOH 

required 

for 

neutral 

izat i on 

of  aci 

dity 

2.7 

3*97 

4, 

12 

4.12 

4.12 

4.12 

4,12 

4.12 

4,12 

3.95 

4. 

45 

4.45 

4.45 

4.45 

4,45 

4.45 

4.45 

5. 

«S5 

3.55 

5*55 

5*55 

5*55 

5*55 

5*55 

5.7O 

5.70 

5*70 

5.70 

5.70 

5*70 

6.10 

6.10 

6.10 

6.10 

6.10 

6.10 

6.10 

6.10 

6.10 

6.30 

6,30 

6.30 

6.15 

6.15 

6.15 

Time 

15’ 

30. 

45 

• 

1 hr 

2 hrs 

3 hr  s 

4 hrs 

8 hrs 

10  hrs 

Standard 

ITaOH 

recxuired 

for 

neutralizati on 

of  acidity 

1.32 

2,70 

3* 

7 

3.82 

3.82 

3.86 

3*85 

3*85 

3*85 

1.90 

3. 

0 

3.15 

3*2 

3.25 

3*3 

3*3 

3*32 

3- 

82 

4 , 32 

4.40 

4.40 

4.5 

4.5 

4.5 

3.87 

4.62 

4.65 

4.65 

4.65 

4.65 

5*0 

5 ..12 

5*12 

5.12 

5 . 12 

5*85 

5.85 

5.83 

5.85 

5*9 

5*9 

5*9 

6,03 

6.03 

-12- 

TABLE  Y-c  ont  inu  ed 


Hydrolysis  of  0.0125  i\r  solution  of  diethyl  adipate  by  lipase. 

25  cc  portions  of  the  ester  solution  were  used.  Ecr  complete 
saponification  of  this  amount  of  ester  3*2  cc  of  0,09872  ]J  KaOH 


were  required , 


Time 

30  * 

1 hr  2 

hrs 

3 hrs  4 

hrs 

5 hrs  6 hrs  8 hrs  9 hrs 

Standard 

NaOH 

required 

f or 

neutralization  of  acidity 

1.425 

2.10 

2.10 

2.15 

2.15 

2.15 

2.15 

2.15 

*6? 

2 7 

2.32 

2 .32 

2,32 

2.32 

2,32 

2.32 

1.05 

2.45 

2.45 

2.45 

2.45 

2.45 

2.4S 

1.85 

2.25 

2.25 

2.25 

2.25 

2.25 

2.1 

2.55 

2.55 

2.55 

2.55 

2.25 

2.35 

2*35 

2.35 

3*075  3*075 

3*35 


-13- 


Ethyl  propionate  .-Data  presented  in  Table  I show  that 
ethyl  propionate  can  be  h3rdrolysed  by  lipase  to  the  extent  of 
90  per  cent  under  the  conditions  of  this  experiment. 

Diethyl  malonate .-The  results  of  the  experiment  with  di- 
ethyl malonate  as  substrate,  Table  II,  are  in  harmony  with 
those  of  Christman  and  Lewis(6)  in  that  they  show  that  the  hy- 
drolysis by  lipase  proceeds  only  half  way  to  completion  under 
the  conditions  of  this  experiment.  A later  experiment  of  a 
different  type  indicates  that  the  extent  of  hydrolysis  can  be 
increased  about  5°  per  cent  by  an  increase  in  the  dilution  of 
the  reaction  mixture,  whether  the  dilution  herein  represented 
by  the  addition  of  6 cc  of  NaOH  or  a 25  per  cent  increase 
is  comparable  to  the  200  per  cent  increase  in  the  later  exper- 
iment would  have  to  be  determined  by  further  study. 

Diethyl  succinate .-The  results  of  the  hydrolysis  of  di- 
ethyl succinate  b3r  liver  lipase,  Table  III,  also  correspond  to 
those  obtained  by  Christman  and  Lewis(6).  An  equilibrium  was 
reached  at  the  end  of  one  or  two  hours  when  the  acid  produced 
by  the  reaction  had  been  neutralized  at  varying  intervals.  The 

rate  of  hydrolysis  increased  up  to  the  point  of  equilibrium 

i,  1; 

and  after  that  time  no  increase  was  noted.  In  both  “7  and  43 


concentrations  of  diethyl  succinate  the  hydrolysis  was  no 
greater  than  50  per  cent  of  the  theoretical. 

1 

Diethyl  glutarate.-The  0.025  N solution  of  diethyl  glutar- 
a.te,  Table  IV,  was  completely  hydrolysed  by  lipase,  while  the 
hydrolysis  of  the  0.05  N solution,  Table  IV, went  only  about 
three  fourths  of  the  way  to  completion.  It  would  also  appear 


-14- 

from  these  data  that  when  the  acid  produced  by  the  reaction  was 
neutralized  an  equilibrium  was  reached  when  the  hydrolysis  had 
proceeded  about  halfway  to  completion  and  beyond  this  point 
hydrolysis  was  very  slight* 

Diethyl  adipate. -The  observation  which  stands  out  most 
prominently  in  regard  to  the  hydrolysis  of  diethyl  adipate, 
Table  V,  is  that  lipase  can  completely  hydrolyse  this  ester. 
From  these  data  it  may  also  be  observed  that  when  the  acidity 
produced  by  the  reaction  was  neutralized  an  equilibrium  was 
reached  after  the  hydrolysis  had  proceeded  two  thirds  of  the 
way  to  completion  and  beyond  this  point  there  was  apparently  no 
further  hydrolysis. 


-15- 


IV  EXPERIMENTAL,  PART  II 

Isolation  of  Products  of  Hydrolysis. 

The  second  type  of  experimentation  was  an  attempt  to  iso- 
late the  products  of  the  hydrolysis  of  the  esters  "by  lipase  . 

In  order  to  isolate  amounts  of  acid  and  ester  sufficient  for 
identification  it  was  necessary  to  ca rvy  out  the  hydrolysis  of 
much  larger  amounts  of  the  esters.  4 cc  portions  of  pure  ester 
were  measured  into  700  cc  Eflenmeyer  flasks.  100  cc  of  HsO 
and  5 cc  of  liver  lipase  were  added.  Two  flasks  were  set  up 
for  each  ester  studied.  Toluene  was  added  as  a preservative 
and  care  was  taken  to  shake  each  flask  frequently  in  order  to 
maintain  an  emulsion  of  the  toluene.  These  flasks  were  incubat- 
ed at  room  temperature  for  a period  of  two  weeks  or  more.  Dur- 
ing this  time  the  acidity'  developed  in  one  flask  was  neutraliz- 
ed daily  while  the  acid  in  the  second  flask  was  not  neutralized 
until  the  end  of  the  period  of  incubation. 

In  order  to  determine  the  extent  of  hydrolysis  of  an  ester 
of  a dicarboxylic  acid  it  is  necessary  to  separate  the  diethyl 
ester  if  any  remains  from  the  monoethyl  ester  and  the  salts. 

At  the  beginning  of  this  investigation  isolation  of  the  products 
of  hydrolysis  was  effected  by  means  of  the  method  of  Christman 
and  Lewis(6).  The  filtrate  from  the  digestion  mixture  was  care- 
fully extracted  several  times  with  ether.  This  should  have  re- 
moved any  unchanged  ester  which  would  have  been  present  in  con- 
siderable quantities  if  the  hydrolysis  of  the  esters  studied 


16- 


had  attacked  both  ethyl  groups  simultaneously.  The  ether  ex- 
tract was  evaporated  at  room  temperature  and  after  solution  of 
the  residue  in  water  aliquots  we re  titrated  to  determine  acid- 
ity and  saponified  to  determine  the  amount  of  saponifiable  mat- 
erial. The  solution  which  remained  after  the  ether  extraction 
was  acidified  with  H2SO4  and  repeatedly  extracted  with  ether. 

The  combined  ether  extracts  were  washed  several  times  and  the 
ether  was  removed  by  evaporation  at  room  temperature.  The  re- 
sidue was  dissolved  in  HaO  and  aliquots  were  titrated  and  sapon- 
ified. This  portion  should  have  contained  the  amid  and  any 
monoethyl  ester  produced  by  hydrolysis.  If  the  hydrolysis  had 
simply  removed  one  ethyl  group  the  amount  of  yw  Ha OH  necessary 
to  neutralize  the  acid  and  to  saponify  the  monoethyl  ester 
should  have  been  equal'.  Table  VI  gives  the  results  of  digest ior 
experiments  in  which  this  method  of  isolation  of  the  products 
was  followed. 


-17- 
TABLE  VI 

The  isolation  of  the  products  of  hydroly 

sis  of  diethyl 

succ inat  e 

and  diethyl 

glutaraoe 

"by  the  method  of  Christman 

and  Lewis. 

Duration  of 

hydrolysis 

17  days.  0.09872 

N NaOH 

ased  for  neutral- 

ization  and 

saponif ica 

t ion. 

Standard 
NaOH  re- 
quired for 
complete 
sap  on  if  ic  ac- 
tion 

Standard 
ITaOH  ac- 
tually re 
quired 

Ether  extract  of  filtrate 

Before  scidi- 
- fication 

After  acidi- 
f ication 

Remarks 

Standard  ITaOH 
to 

Standard 

to 

NaOH 

Neutral-  Sap- 
ize 

Neutral- 

ise 

Sap- 

onify 

231 

105.4 

None  1.88 

20.0 

23.I 

2 c c of 
diethyl 
succinate 
neutral  is 
ed  at  end 

231 

126.2 

None  1,12 

28.0 

28.8 

2 cc  of 
diethyl 
succinate 
neutraliz- 
ed dally 

463 

144,4 

None  None 

18.8 

24.32 

4 cc  of 
diethyl 
succinate, 
neutraliz- 
ed at  end 

463 

231.75 

None  None 

68.8 

69.44 

4 cc  of 
diethyl 
succinate 
neut raliz- 
ed  dally 

430 

149.2 

None  14.4 

32.48 

44.2 

4 c c of  d i 
diethyl 
glutarate 
neutraliz- 
ed at  1 

430 

309*1 

None  25.2 

41,20 

40,0 

4 cc  of-" 
diethyl 
6 lu tar ate 
neut raliz- 

430 

257*8 

None  1.2 

45*6 

47.2 

4 cc  of 
diethyl 
glut a rat e 
neutraliz- 
ed daily 

-18- 

Diethyl  succinate, -The  data  on  Table  VI  are  of  value  in 
that  they  show  the  extent  hydrolysis  of  the  esters  under  dif- 
ferent conditions  of  acidity*  The  data  regarding  the  products 
of  the  hydrolysis  point  to  inaccuracies  in  the  recovery  of  acid 
and  ester.  If  lipase  can  remove  only  one  ethyl  group  from  di- 
ethyl succinate  then  the  reaction  represented  which  was  neutral- 
ized daily  had  gone  to  completion.  If  both  ethyl  groups  can  be 
removed  by  the  lipase  the  same  reaction  has  gone  only  half  way 
to  completion.  This  fact  cannot  be  determined  from  these  data. 
Th@  retardation  of  the  hydrolysis  reaction  when  the  acid  produc- 
ed was  not  neutralized  may  have  been  due  to  the  concentration 
of  the  solution.  Later  experiments  of  the  same  type  seem  to 
show  that  hydrolysis  proceeds  farther  when  K2O  is  added  daily, 

Diethyl  glutarate.-It  may  be  observed  that  under  the  condi- 
tions of  this  type  of  experiment  the  hydrolysis  of  diethyl  glu- 
tarate  proceeded  only  about  half  way  to  completion  even  when  the 
acid  produced  was  neutralized  frequently.  The  figures  represent- 
ing the  daily  titrations  show  that  the  hydrolysis  proceeded  very 
rapidly  at  first  and  that  an  equilibrium  was  approximately  reach- 
ed at  the  end  of  the  third  day.  It  appears  that  when  the  acid 
produced  by  the  react! 021  was  neutralised  the  hydrolysis  readily 
proceeded  half  way  to  completion  while  after  that  point  was 
reached  the  hydrolysis  proceeded  relatively  slowly. 

As  the  investigation  progressed  certain  errors  were  suspect- 
ed and  therefore  modifications  were  made  in  the  method  of  re- 
covery. TablesVII  and  VIII  give  the  results  of  the  hydrolysis 


- 


- 


, 


. 


-19- 

of  diethyl  succinate  and  the  products  isolated  noy  modifications 
of  the  method  of  Christiaan  and  Lewis(6)» 


“20- 

TABLE  VII 

Isolation  of  the  products  of  hydrolysis  of  4 cc  of  ethyl  suc- 
cinite by  a slight  modification  of  the  method  of  Christman  and 
Lewis  which  consisted  in  salting  cut  with  (NIU  )aS04  before  ex- 
traction with  ether.  Duration  of  hydrolysis  40  days.  0.09872 
N NaOH  used  for  neutralization  and  saponification. 


Standard 
NaOH  re- 
quired for 

Standard 
NaOH  ac- 
tually 

Ether  ex 

tract  c 

>f  Filtrate 

Before  acid- 
ification 

After  acid- 
ification 

Remarks 

complete 
saponif i- 
c at ion 

required 

Standard 

to 

NaOH 

Standard 

to 

NaOH 

Neutral- 

ise 

Sap- 

onify 

Neutral- 

ize 

Sap- 

onify 

463 

231 

None 

1.08 

76.8 

?8,8 

Neutral- 
ised daily 

463 

165 

None 

14.4 

61.6 

6l,6 

Neutral- 
ized at  end 

-21- 

TABLE  VIII 

Isolation  of  the  products  of  hydrolysis  of  4 cc  of  diethyl 
adipate  by  a modification  of  the  method  of  Christman  and  Lewis 
(6).  This  modification  consisted  in  evaporating  the  filtrate 
to  dryness  before  extraction  which  was  done  with  warmed  ether. 
Duration  of  hydrolysis  IS  days.  0.098/2  N La OH  was  used  for 
neutralisation  and  saponification. 

Standard 
HaOH  re- 
quired for 
complete 
saponifi- 
cation 

Standard 
La OH  ac- 
tually 
required 

Ether  Extract  of  filtrate 

Remarks 

Before  acid- 
. ification 

After  acid- 
ification 

Standard  NaOE 
to 

neutral- 

ise 

Sap- 

onify 

400.1 

401.2 

Hone 

Hone 

Contained  a 
quantity  of 
white  cr3T- 
stals  LLP. 
14?. 6°  H.E. 
?6  and  no 
saponiif ia- 
ble  material 

neutralized 

daily 

400.1 

152.5 

Hone 

Hone 

Contained  a 
quantity  of 
white  cry- 
stals. M.P, 
146°  and  sap- 
onifiable mat- 
erial equiva-v 
lent  to  6 
HaOH 

Neutralised 
at  end 

400.1 

209 

4.0 

Hone 

Contained  a 
quantity  of 
white  cry- 
stals. M.  P. 
147  and  sapon- 
ifiable mater- 
ial equivalent 
to  34.4  cc  — £ 
HaOH 

Neutralised 
daily  and  re- 
action stop- 
ped when  hydro 
l3rsis  he.d  gone 
half  way  1 0 
complexion 

-22- 


Results  presented  in  Table  VIII  are  of  interest  in  as  much 
as  they  show  that  diethyl  adipate  may  be  completely  hydrolysed 
b3T  liver  lipase  when  the  acidity  produced  by  the  reaction  is 
neutralized  daily*.  The  fact  that  the  hydrolysis  was  greatly  re- 
tarded when  the  acid  produced  by  the  reaction  was  not  neutral- 
ized may  have  been  due  to  the  concentration  of  trie  solution.  It 
is  interesting  to  note  that  when  the  hydrolysis  of  4 cc  of  di- 
ethyl adipate  had  proceeded  half  way  to  completion  as  indicated 
by  the  amount  of  standard  HaOH  which  had  been  used  to  neutralize 
the  acid  produced  cryst  als  of  adipic  acid  were  obtained  which 
shows  that  the  second  ethyl  group  was  attacked  before  the  first 
had  been  entirely  removed* 

Since  the,  theoretical  yield  of  acid  or  ester  had  not  been 
obtained  by  ary  modification  of  the  method  of  Lewis  and 
Christman( 6 ) it  was  decided  to  try  tc  recover  acid  and  ester 
from  known  amounts  of  each  under  the  same  conditions  as  existed 
J ^ ^ - digestions . Table  IX  contains  the  results  of  these 

trials.  17  o quantitative  recovery  of  either  acid  or  ester  was 


i 


obtained , 


It  was  thought  that  the  solid  portions  of  the  enzyme  ex- 
tract  may  ha.,  o adsorbed  some  of  the  ester  and  therefore  if  fil- 
tration could  be  omitted  one  source  of  error  would  be  eliminat- 
ed. It  also  seemed  advisable  to  eliminate  the  second  ether  ex- 
-ractioii.  To  make  this  possible  the  solution  from  the  first 
ef.aer  extraction  was  acidified  with  an  excess  of  HC1  and  evapor- 
ated lc  dryness  on  the  steam  bath.  The  residue  was  dissolved  in 


-23- 


HaO  and  aliquots  wer  titrated  and  saponified  with  stand trd 
itfaOH  to  determine  the  amount  of  dicarboxylic  acid  and  monoeth 
ester  present. 

The  next  thought  that  suggested  itself  was  the  use  of 
CHCls  as  a solvent  in  place  of  ether.  Tables  X,  XI,  and  XIT 
give  the  results  of  some  preliminary  experiments  carried  out 
in  an  effort  to  find  a suitable  method  of  isolation. 


<.A 


-24- 
TABLE  IX 

The  isolation  of  succinic  acid  and  succinic  ester  from  a neu- 
tralized solution  containing  2.4  grams  of  the  acid  and  1.6  grams 
of  the  ester  by  means  of  salting  out  with  (HIU)2S04  and  extract- 
ing with  ether  before  and  after  acidification. 

2.4  grains  of  succinic  acid  are  equivalent  to  410.  3 cc  O.09S72  H 
Ha  OH 

1.6  grams  of  succinic  ester  are  equivalent  to  182  cc  0,09872  H 


IT  a OH 


Number 

of 

Exper- 

iment 

Ether  Extract 

of  Solution 

B ef  or  e ac  id  if  i c at  i on 

After  acidi: 

f ication 

Standard  Ha 0E  to 

Standard  Ha  OH  to 

__  Neutralize 

Saponify 

Neutralize  I 

y 

at. 

Hone 

48  cc 

2,4 

Hone 

II 

Hone 

29.2 

15.0 

18 

V.  - 


_0  £ „ 

CL.sJ 


Solutions  of 
with  CKCls . 
of  0.098^2  IT 


TAB  IT3;  XT 

diethyl  succinate  and  succinic  acid  were  extracted 
1 gram  of  succinic  acid  is  equivalent  to  171  cc 
ITaOH.  1 gram  of  diethyl  succinate  is  equivalent 


to  114  cc  of  O.O8872  N NaOH. 


Number 

of 

Exp  er- 

iment 

TT30  Extraction 

CKCls  Extraction 

Remarks 

Standard  ITaOH  to 

Standard  NaOH  to 

— 

Neutral- 

ise 

Sapon- 

ify 

Neutral- 

ize 

Sap  on- 
5 f 7 

T 

280.0 

188.0 

None 

16.0 

1 cc  diethyl 
succinate 
1.5  grams  suc- 
cinic acid 

II 

185 . 0 

102.0 

Lost 

Lost 

1 cc  ethyl  suc- 
c inat e 

1 gr . succinic 
acid 

III 

236.O 

214.0 

2 cc 

51.0 

2 cc  diethyl 
succ inate 
1.5  grams  suc- 
cinic acid 

-28- 

As  is  apparent  from  a reading  of  the  data  given  these 
methods  of  isolation  had  not  proven  satisfactory.  The  greater 
acidity  obtained  when  the  filtrate  was  acidified  with  HC1  and 
evaporated  to  dryness  may  have  been  due  to  some  residual  HC1. 

The  larger  amounts  of  saponifiable  material  obtained  in  the  H3O 
soluble  portion  when  chloroform  was  used  as  a solvent  may  have 
been  due  to  decomposition  of  some  dissolved  chloroform  in  alka- 
line reaction.  It  was  also  thought  that  during  evaporation  of 
the  chloroform  on  the  steam  bath  steam  distillation  of  the 
ester  may  have  taken  place. 

It  seemed  worth  while  to  try  vacuum  distillation  as  a means 

of  separating  the  partially  hydrolysed  from  the  unhydrolysed 

ester* Preliminary  experiments  using  solutions  of  ester  and  acid 

were  carried  out  which  proved  that  any  diethyl  ester  present  in 
solution 

a neutral /was  carried  over  almost  quantitatively  in  the  distil- 
late and  that  the  monoethyl  ester  and  the  salts  were  left  in 
the  residue  apparently  unchanged.  In  an  attempt  to  obtain  the 
acid  and  monoethyl  ester,  the  residue  was  acidified  and  extract- 
ed with  ether.  Tables  XTTI  and  XIV  give  the  data  obtained  from 
the  preliminary  experiments  and  Table  XV  the  data  ol  • ined  from 
the  use  of  this  method  in  the  isolation  of  the  products  of  a 
digestion  b3r  lipase. 


-30- 

TABLE  XIV 

One  gram  of  diethyl  succinate  and  one  gram  of  succinic  acid  and 
5 cc  of  boiled  enzyme  were  neutralized  aud  distilled  in  vacuo. 
The  residue  was  acidified  and  extracted  with  ether. 

1 gran  of  diethyl  succinate  is  equivalent  to  114  cc  0. 09872  IT 

Ha  OH 

1 gram  of  succinic  acid  is  equivalent  to  171  cc  O.O9872  H Ha OH 


Distillate 

Ether  Extract  of  Residue 

Standard  Ha OH  to 

Standard  Ha OH  to 

neutralize 

Saponify 

neutralize 

Saponify 

Hone 

93. 8 

Lost 

' Lost 

One  gram  of  potassium  ethyl  malonate  and  one  gram  of  malonic 
ester  and  3 cc  of  boiled  enzyme  were  dissolved  in  HaO  and  dis- 
tilled in  vacuo.  The  residue  was  acidified  and  extracted  with 

ether . 

1 gram  of  potassium  ethyl  malonate  is  equivalent  to  58.8  cc 
0,09872  H Ha OH 

1 gram  of  malonic  ester  is  equivalent  to  150  cc  of  0. 09872  N HaO?) 


Distillate 

Ether  Extract  of  Residue 

Standard  Ha OH  to 

Standard  Ha  OH  to 

neutralize 

Saponify 

neutralize 

Saponify 

Hone 

144.0 

4 cc 

32.0 

- 

1 1 


. 


. 


* 


-31- 

table  XV 

Isolation  of  products  of  hydrolysis  of  diethyl  malonate  and 
diethyl  succinate  hy  distillation  in  vacuo  and  ether  extraction 
of  acidified  residue.  Duration  of  hydrolysis  3°  days.  O.Q9872 


IT  ITaOH  was  used  for  titration  and  saponification. 


Standard 
ITaOH  re- 
quired for 
complete 
sap on if i ca- 
tion 

Standard 
NaOH  ac- 
tual lv 
required 

Diethyl 
ester  in 
equiva- 
lents of 

0.09872 

IT  ITaOH 

Ether  Extract  of 
Ac  id if i ed  He  s idue 

Remarks 

Standard 
ITaOH  to 
neutral- 

ire  . 

Standard 
ITaOH  to 
Baponi— 

L_£sl.  , , 

510 

300 

4.0 

8.0 

26.0 

4 cc  diethyl 
malonate 
neutral ized 
daily 

510 

120 

93.0 

Undetermined 

4 cc  diethyl 
malonate 
H2O  added 
daily  to  keep 
volume  equal 
to  neutraliz- 
ed mixture 
neutralized 
a t end 

510 

340 

12.0 

2 0.0 

30. c 

4 cc  diethyl 
malonate  used 

5 cc  lipase 
extract  added 
when  equili- 
brium reached 
neutralized 
daily 

463 

300 

3.0 

128.0 

102.0 

4 cc  diethyl 
succinate 
neutral ized 
daily 

4^3 

230 

66 . 0 

Undet g 

s mined 

4 cc  diethyl 
succinate 
F2O  added 
daily  to  keep 
volume  equal 
'gg  that  of 
neutralized 
mixture .neu- 
tralized at 
end . 

-32- 


V DULMARY 


I«A  study  of  the  hydrolysis  of  the  diethyl  est 
the  dicarboxylic  acids  'ey  liver  1 ij:  ' 

of  hydrolysis  changes  with  a.,  increase,  in  the  molecular  weight 
of  bhe  ester.  Under  the  sane  experimental  conditions  lipase 
app  n\  removed  both  ethyl  groups  from  diethyl  adipate  an 
diethyl  glutarate  while  the  hydrolysis  of  diethyl  r.aicnat e and 
of  diethyl  succinate  progressed  only  half  way  to  completion. 

II. * Crystals  of  pure  adipic  acid  and  no  saponifiable 
material  were  obtained  from  the  hydrolysis  of  diethyl  adipate 
cy  liver  lipase  when  the  acid  produced  by  the  reaction  was  neu- 
tralized daily. 

III.  men  the  hydrolysis  of  diethyl  adipate  by  liver 
lipase  was  stopped  half  way  in  its  progress,  crystals  of  pure 
adipic  acid  were  obtained  as  one  of  the  products  of  the  hydro— 

lysis* 

J 'v  * ;-n  cases  when  4 cc  of  ester  was  incubated  with 
liver  lipase  for  a period  of  time  the  hydrolysis  progressed 
farther  wren  me  acid,  produced  by  the  reaction  was  neutralized 

daily . 

■ . u all  cases  when  4 cc  of  ester  was  incubated  with 
liver  lipase  over  a period  of  time  and  the  acid  produced  by  the 
reaction  wcm  not  neutralized  daily  the  extent  of  the  hydrolysis 
was  greater  when  HsO  was  added  daily  than  under  otherwise  simi- 
lar conditions. 

VI. The  addition  of  5 cc  of  enzyme  to  a reaction  mixture 


. 


-33- 

which  had  apparently  reached  an  equilibrium  had  very  little  ef- 
fect  on  the  extension  of  the  hydrolysis* 

VII*With  the  exception  of  the  experiments  with  diethyl 
adipate  complete  recovery  of  the  products  of  hydrolysis  - as  not 
attained  however,  while  the  results  of  the  methods  employed  in 
general  point  to  the  conclusions  of  Lewis  and  Christman  regard- 
ing the  hydrolysis  of  diethyl  maloriate  and  diethyl  succinate 
they  also  open  up  various  possibilities  of  research. 


